NEW YORK (Genome Web) – In a bid to standardize workflows for microbiome studies, researchers at Human Longevity have compared different microbiome sample stabilization approaches.
"It's a big issue that the community cannot compare samples," Human Longevity's Karen Nelson told GenomeWeb.
Nelson and her colleagues compared how human fecal samples stabilized using DNA Genotek's Omnigene Gut kit stacked up against fresh and frozen samples in terms of nucleic acid yield and reproducibility. As they reported in Scientific Reports this week, the researchers found that samples stabilized using the Omnigene Gut kit performed just as well as frozen ones and even yielded higher amounts of nucleic acids.
In its paper, the HLI team noted that the validity and reproducibility of microbiome studies hinge on the quality of the samples used and that stabilization and proper storage of those samples is a fundamental step in obtaining a clear look at the microbiome.
The Omnigene Gut kit stabilizes microbial DNA from fecal samples for storage and analysis. According to DNA Genotek's Aaron Del Duca, vice president of technology and head of the company's microbiome business unit, the buffer used in the kit prevents enzymatic and chemical degradation of the samples and includes antimicrobials to avoid overgrowth. The samples are homogenized and can be stored as liquids at room temperature for weeks.
The kit is available in the US for research use and has a CE mark for in vitro diagnostic use in Europe.
Nelson and her colleagues compared samples stabilized using this approach to frozen — the current standard recommended by the Human Microbiome Project and the Centers for Disease Control and Prevention — and fresh samples collected from 16 volunteers. DNA and RNA were then extracted from each for metagenomic sequencing on the Illumina HiSeq 2500. HLI's Ericka Anderson noted that they chose to evaluate this kit rather than other reagent mixes for stabilization, as it was on the market as a full kit when they began their study, it provided a fixed amount of space for the sample, and homogenized the samples at the time of collection.
Overall, they and their colleagues reported that Omnigene Gut kit-stabilized samples were largely comparable to samples frozen to -20° Celsius. In particular, the researchers found that their assembly and sequencing metrics weren't affected by the stabilization approach and that the taxonomic and abundance profiles of stabilized samples mostly corresponded to those of basal and frozen samples. Intra-subject variation, they reported, was greater than that due to the stabilization approach used.
In addition, they found that both stabilized and frozen samples yielded reproducible results. That is, the relative species abundance values they noted for them on day 0, day 1, and day 28 were consistent for most samples for both approaches. They likewise had similar species richness and diversity measures.
Stabilization using the Omnigene Gut kit did yield higher quantities of DNA and RNA than other approaches. DNA Genotek's De Duca said this could be due to the buffer's ability to halt enzymatic and chemical breakdown of nucleic acids, and HLI's Anderson added that the early homogenization step might enable the buffer to be more evenly distributed around the sample, noting that they haven't tested this idea. At the same time, De Duca noted that freezing cracks samples open, exposing them to stool enzymes that could chew up nucleic acids.
Further, as these samples are stored at ambient temperature, this stabilization approach can be used in areas where freezing isn't readily available, Nelson said. That, she added, would enable more global samples to be collected for analysis.
At the same time, Anderson said it eliminates the freeze-thaw cycle that can affect sample quality over time. And, she said, it's cheaper. She and her colleagues noted in their paper that this approach eliminates the need for freezers as well as for dry ice and cold pack shipping.
Still, freezing is the currently accepted standard in the community. "I'd love for it to change, but it's hard to predict," Anderson said. "The data [indicate] these stabilization reagents do as well as or better than freezing, and when you take into account all the costs … it's cheaper."
The HLI investigators previously compared different library preparation products, and in this study moved on to gauge the best approach for another part of their microbiome analysis pipeline. Nelson added that her group at HLI would continue to evaluate new tools as they become available and would examine other parts of their pipeline, including informatics.